Method of determining cholesterol

ABSTRACT

Blood serum containing cholesterol is treated with water and a reagent containing glacial acetic acid, acetic anhydride, and sulfuric acid to form a pink colored complex whose absorbance is proportional to the cholesterol concentration. The amount of cholesterol can be determined by measuring the optical density or absorbance of the colored complex at 500 nm. In order to produce the colored complex and prevent interference, for each unit volume of blood serum, 15-24 unit volumes of water and 200 unit volumes of reagent are utilized. The preferred ratio of the volumes of serum to water to reagent is 1 to 19 to 200.

United States Patent Dixon et al.

ADD CHOLESTEROL COLOR REAGENT METHOD OF DETERMINING CHOLESTEROLInventors: Cheryl Dixon, Walpole; Arnold Alpert, Natick, both of Mass.

Damon Corporation, Needham Heights, Mass.

Filed: Aug. 1, 1973 Appl. No; 384,515

Assignee:

US. Cl. 23/230 B Int. Cl. G0ln 33/16 Field of Search 23/230 B; 252/408References Cited UNITED STATES PATENTS 11/1969 Cardinal 23/230 B 2/1973Denny 23/230 B ADD SAMPLE 2 3,733,179 5/1973 Guehler 23/230 B PrimaryExaminer-R. E. Serwin Attorney, Agent, or Firm-Kenway & .lenney [5 7ABSTRACT Blood serum containing cholesterol is treated with water and areagent containing glacial acetic acid, acetic anhydride, and sulfuricacid to form a pink colored complex whose absorbance is proportional tothe cholesterol concentration. The amount of cholesterol can bedetermined by measuring the optical density or absorbance of the coloredcomplex at 500 nm. In order to produce the colored complex and preventinterference, for each unit volume of blood serum, 15-24 unit volumes ofwater and 200 unit volumes of reagent are utilized. The preferred ratioof the volumes of serum to water to reagent is l to l9 to 200.

9 Claims, 1 Drawing Figure 0 10 TO PHOTOMETER TEST PATENTED W? 9 5 I"/"DO, ADD SAMPLE f 3 ADD CHOLESTEROL COLOR REAGENT METHOD OFDETERMINING CHOLESTEROL BACKGROUND OF THE INVENTION The field of thepresent invention is an analytical procedure for quantitativelydetermining the amount of cholesterol in a sample to be analyzed.

Cholesterol, either in the free form or as an ester, is an essentialconstituent of all animal and plant cells, and is often accompanied byits derivatives, dihydrocholesterol and 7-dihydrocholesterol. Bloodcholesterol, both esterified and free, is one of the major blood lipidsand is present together with total fatty acids, triglycerides, andphosphatides. Cholesterol in the blood is distributed almost equallybetween the plasma and corpuscles, with higher concentrations in theleukocytes. Human blood plasma normally contains 100 to 320 mg. of totalcholesterol, with a mean of 160 mg. per 100 cc. of plasma which isusually so distributed that about one-fourth is free cholesterol and theremaining three-fourths is the cholesterol ester of fatty acids. Sincethe sum of the free cholesterol and the cholesterol esters in the serumis usually referred to as total cholesterol, for convenience, this termwill be so applied hereinafter.

Every human being maintains a constant total serum cholesterol level andlarge deviations from this level do not ordinarily occur. However,research directed to the effects of abnormally high total cholesterolblood levels has made it increasingly apparent that the totalcholesterol blood level should be controlled and should be maintainedwithin the limits considered normal for the particular individual. Thetotal cholesterol level in the blood plasma tends to be high, forexample, in hyperglycemia and in uncontrolled diabetes. Cholesterol hasalso been found to be the principal lipid in the intima of the arterialwall in atherosclerosis. Plasma cholesterol levels are also high inicterus, naphritis, alcoholism, anesthesia, pregnancy, syphilis, andfollowing excision of the adrenals. Total serum cholesterol is alsoelevated in diseases of the liver associated with obstruction of bileflow, in familial hypercholesterolemia, in hypothyroidism and in thenephrotic syndrome. Low blood cholesterol is found in severe liverdisease, wasting illness, malnutrition, and'hyperthyroidism.Accordingly, the accurate determination of serum cholesterol levels isimportant for modern diagnostic procedures.

Numerous reagents have been employed in methods for the determination oftotal cholesterol in biological fluids such as serum or plasma or thelike. In all of these methods, the amount of total cholesterol in asample of a biological fluid is normally determined by contacting thebiological fluid with a reagent which combines with cholesterol in thefluid to form a colored reaction product. The reagent is mixed with thefluid in such a way that the depth or intensity of color produced isproportional to the amount of cholesterol present in the sample ofbiological fluid. The concentration of cholesterol in the sample is thendetermined by measuring the depth or intensity of color, usually with acolorimeter or a spectrophotometer. By use of conversion charts orcomparisons to standard solutions, the measurement of the color producedby the use of the reagent can be converted to give the concentration oftotal cholesterol in the sample.

Among the most widely accepted methods typical of the prior artdeterminations is the method of Abell et al., J. Biol. Chem., 195, 357(1952). In the method of Abell et al., the serum sample is first treatedwith alcoholic potassium hydroxide to liberate cholesterol fromlipoprotein complexes and saponify cholesterol esters. The sample isthen extracted with a predetermined volume of petroleum ether. The etherextract is evaporated and the residue is then treated with a reagentcomprising sulfuric acid, glacial acetic acid and acetic anhydride toform a colored product. The intensity of color produced is then measuredin a colorimeter or spectrophotometer to give a value which isproportional to the concentration of cholesterol in the sample.

The saponification and extraction steps make this procedure lengthy andtime-consuming. Direct methods employing the same reagent have reducedthe time required for analysis, but have necessitated a correction ofthe results obtained to account for interference caused by the presenceof bilirubin in the sample. The correction depends on the amount ofbilirubin present in the sample. Also, the reagent employed is notstable in storage; thus, frequent preparation of fresh reagent has beennecessary.

Various direct methods have employed a reagent comprisingp-toluenesulfonic acid, acetic anhydride, glacial acetic acid andsulfuric acid. See, for example, Van Boetzelaar and Zondag, Clin. Chim.Acta, 5,943 (1960). The methods employing p-toluenesulfonic acid haverequired corrections for bilirubin. Other reagents have been employed,including a mixture offerric chloride, sulfuric acid and glacial aceticacid as described by Zlatkis et al., J. Lab. Clin. Med, 41, 486 1953)and Furst et al., Scand J. Clin. Lab. Invest, 6, 60 (1954).

A significant drawback associated with prior art direct measurementsutilizing glacial acetic acid, acetic anhydride and sulfuric acid isthat the water used to dilute a serum sample interferes with theanalysis. In connection with the foregoing, it should be noted thatautomated blood analyzers normally analyze blood serum that has beendiluted with water before the analysis.

SUMMARY OF THE INVENTION In accordance with the present invention, waterand a reagent including glacial acetic acid, acetic anhydride andsulfuric acid is reacted with serum containing cholesterol to determinethe amount of cholesterol present therein. In order to preventinterference and produce a complex which can be measured at 500 nm, theratio of serum to water to reagent is maintained within the range of oneunit volume of serum to 15-24 of water to 200 of reagent.

Accordingly, it is an object of the present invention to provide a newand improved method for direct determinations of the amount ofcholesterol in blood serum.

A further object of the present invention is to provide a method fordirectly measuring the amount of cholesterol in blood serum utilizing areagent which includes glacial acetic acid, acetic anhydride andsulfuric acid in which interfering reactions are eliminated.

A further object of the present invention is to provide a method fordetermining the amount of cholesterol present in blood serum which hasbeen diluted with water in which the water is a reacting material whichcontributes to the development of a chromogen.

A further object of the present invention is to provide a new andimproved method for the measurement of cholesterol which can be utilizedin any automated systern in which a dilution of a sample with wateroccurs prior to the analysis.

BRIEF DESCRIPTION OF THE DRAWING The sole FIGURE of the drawing is adiagram illustrating the operating parameters of the process of thepresent invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS At the outset, the process ofthe present invention is described in its broadest overall aspects witha more detailed description following. In the method of the presentinvention, cholesterol is treated with water and a reagent includingglacial acetic acid, acetic anhydride and sulfuric acid to form coloredsubstances. Most probably, the colored substances are composed ofbischolestadiene disulfonic acids. The colored substances have a pinkcolor, are proportional to cholesterol concentration and are measured at500 nm.

One important factor that enables the formation of the pink coloredsubstances is the amount of water that is present in the system. Inaccordance with the present invention, for each unit of volume of serumto by analyzed, -24 unit volumes of water to 200 unit volumes of reagentare utilized. The optimum volume ratio of serum to water to reagent is lto 19 to 200.

The method of the present invention can be performed manually. Thedeterminations of appropriate parameters such as volume measurements,timing and temperature controls are well within the skill of those inthis art. The preferred embodiment of the present invention, however, isperformed in an automatic blood analyzer such as the type manufacturedby Damon Corporation, Needham Heights, Massachusetts, under the nameAcuChem Microanalyzer. Details of this type of automatic blood analyzerare set forth in US. application Ser. No. 191,884 entitled Constituents-Measuring Chemical Analyzer having Sample- Processing Conduit FeedingAliquot-Processing Conveyor System by David I. Kosowsky, Andres Ferrariand Carl R. I-Iurtig, filed Oct. 22, 1971, the teachings of which areincorporated herein by reference.

The analytical process of this type of blood analyzer is illustrated bythe operating parameter diagram set forth in the sole FIGURE of thedrawing. The parameter diagram shows a circular layout of paired circleswhich forms a probe position plan for a given method of analysis. Eachpair of circles represents the one or two positions in each segment of aprobe support plate available for each method of analysis on the AcuChem.Microanalyzer. On the plate itself, corresponding positions in eachsucceeding segment are reserved for that same method. The parameterdiagram shows the function assigned for the methods as the test tubespass under the probe support plate. In one important embodiment of theinvention, prior to running an analysis with the AcuChem Microanalyzer,the instrument is calibrated with calibrating fluids. The calibratingfluids contain a known amount of the constituent of blood serum forwhich an analysis is sought. For example, when cholesterol is to beanalyzed, the AcuChem instrument is calibrated by placing a known amountof cholesterol in the sample and analyzing that sample. The preferredmethod of calibrating the AcuChem Micro-analyzer is to utilize severalsamples of known constituents. Typically, the machine is calibrated withconstituents of blood serum in three ranges. The ranges are selected onthe basis of a low amount, a mid-range amount and a high amount of theparticular constituent as compared to that amount which would be presentin normal blood serum. At this point, it should be noted that theAcuChem instrument is capable of performing an analysis for more thanone constituent of blood. Thus, the calibrating fluids contain more thanone blood serum constituent. In effect, the calibrating fluids areprepared in the laboratory and contain all those constituents normallypresent in blood serum which are to be analyzed in the AcuChemMicroanalyzer.

The AcuChem Microanalyzer will process samples at a rate of 60 per hour.All splits from a single sample, having been diluted 1:20, aresimultaneously introduced through probes in segment No. 1 (see the soleFIGURE of the drawing) of the probe support plate into test tubes in thetest tube plate. Each segment of the test tube plate, containing dilutedserum portions from one patient, passes under the probe support plate atthe rate of one segment per minute. Thus, the various functions ofreagent dispensing, mixing, transfer to photometer, washing and dryingare carried out in turn according to the probe position plan. Theinstrument design allows nine segments between sample addition andtransfer to photometer; hence, a 10 minute dwell time. Thus, ninesegments are available for carrying out the required steps in aparticular chemistry. After the reacted sample has been transferred tothe photometer and while the test tubes are still under segment No. 11,wash water is added. This is then aspirated and functions as a flow cellwash. Additional washing and drying of the test tubes is carried out asthey pass under segments No. 12 No. 24.

The process of the present invention is further illustrated by thefollowing non-limiting examples.

EXAMPLE 1 Automatic Determination of Cholesterol A sample of blood serumhaving been diluted with deionized water to yield a water-to-serum ratioof 20 to 1 is introduced into segment No. 1 of an AcuChem Microanalyzer.At segment No. 2, cholesterol color reagent is added to the sample. Atsegment No. 3, the reagent and sample are mixed. At segment No. 11, theabsorbance of the sample at 500 nm is measured by a photometer. Thecholesterol color reagent added at segment No. 2 is prepared as follows:Cholesterol-Reagent Preparation To make 1,000 ml. of cholesterol colorreagent, 483 ml. of acetic anhydride (Baker No. 0018) is added to 276ml. of glacial acetic acid (aldehyde-free, Baker No. 951 1) withcontinuous mixing and cooling. To this mixture (while mixing and coolingcontinue), 241 ml. of concentrated sulfuric acid (H SO Fisher No.A-300C) is added slowly so as not to allow the temperature to rise above30C. The reagent is stored in amber glass or polyethylene bottle.

Various operating parameters for this analysis are given below.

Path length 50 mm Sample or Reagent Vol. (ul) Segment 1. Sample-Continued Cholesterol color reagent Segment 2. 1000 EXAMPLE 2Cholesterol-Nonautomated (Manual) Method 1. Using deionized water as thediluent, three 1:20 dilutions of calibration fluids containing knownamounts of cholesterol in a low, mid and high range are prepared.

2. 1:20 dilutions of the unknown serums using deionized water as thediluent are prepared.

3. To 0.20 ml. of deionized water, 2.0 ml. of the cholesterol colorreagent as prepared in Example 1 is added and mixed well. This is thereagent blank.

4. To 0.20 ml. of diluted unknowns or calibration fluids, 2.0 ml. of thecholesterol color reagent is added and mixed well.

5. 8 to 12 minutes after mixing the water and cholesterol reagent, themixture is poured into a mm light path cuvet and a photometer isadjusted (set at 500 nm) to zero.

6. Eight to twelve minutes after mixing diluted unknowns or calibrationfluids and cholesterol reagent, the mixture is poured into a 10 mm lightpath cuvet and the absorbance is read.

7. With good quality graph paper, absorbances of the reacted calibrationfluids is plotted on the ordinate versus their cholesterolconcentrations on the abcissa.

8. Referring to the calibration curve, concentrations of unknowns isread off directly in mg/dl cholesterol.

Note: Unknowns and calibration fluids should be assayed in duplicate andthe mean of the duplicates should be used for final preparation and useof the calibration curve.

Accordingly, by following the teachings of the present invention, a newand improved method for the quantitative analysis of cholesterolresults. By controlling the serum to water to reagent ratio inaccordance with the present invention, it is possible to measurecholesterol concentration directly without making any interferenceadjustments. In the prior art methods, when a cholesterol color reagentwhich included glacial acetic acid, acetic anhydride and sulfuric acidwas utilized, interference adjustments were necessary.- However, inaccordance with the present invention, water in the proper proportioncontributes toward the development of the chromogen and there is nosignificant interference. Furthermore, the process of the presentinvention produces a chromogen which is different from the prior artchromogen which occur with the same reagent, i.e., glacial acetic acid,acetic anhydride and sulfuric acid. in this regard, the process of thepresent invention is broadly directed to maintaining a proper ratio ofserum to water to reagent to produce a pink substance which absorbslight in wavebands centered at 500 nm. Prior art processes utilizingglacial acetic acid, acetic anhydride and sulfuric acid as a colorreagent produced a blue-green substance which absorbed light withwavebands centered between the range of 600 650 nm.

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

We claim:

1. A method for determining total cholesterol comprising treating thecholesterol with water and a reagent containing acetic acid, aceticanhydride and sulfuric acid to form a colored substance which absorbslight at 500 nm, and measuring the optical density of the coloredsubstance.

2. In a method for determining the amount of cholesterol in blood serumin which blood serum is treated with a reagent comprising sulfuric acid,acetic acid and acetic anhydride to form a colored product and theintensity of the color produced is measured to give a value which isproportional to the concentration of the cholesterol in the blood serum,wherein the improvement comprises diluting the blood serum with waterand maintaining the ratio of blood serum to water to reagent within therange of one unit volume of blood serum to 15-24 unit volumes of waterto 200 unit volumes of reagent.

3. The method as set forth in claim 2 including the step of measuringthe absorbance of the color produced formed at 500 nm.

4. The method as set forth in claim 3 wherein the ratio of serum towater to reagent is 1 to 19 to 200.

5. A method for determining the amount of cholesterol in blood serumcomprising diluting the blood serum with water, treating the blood serumwith a reagent comprising sulfuric acid, acetic acid and aceticanhydride to form a colored substance which absorbs light at 500 nm,thereafter measuring the intensity of the colored substance at 500 nm.

6. The method as set forth in claim 5 wherein the ratio of serum towater to reagent is maintained within the range of 1 to 15-24 to 200.

7. The method as set forth in claim 5 wherein the serum is diluted withwater to have a serum to water ratio of l to 20.

8. The method as set forth in claim 7 wherein 1,000 ml of the reagentcontains approximately 483 ml of acetic anhydride, 276 m1 of acetic acidand 241 m1 of sulfuric acid.

9. The method as set forth in claim 8 wherein each 11.1 of blood serumand water is treated with 1,000

11.1 of reagent.

1. A METHOD FOR DETERMINING TOTAL CHOLESTEROL COMPRISING TREATING THECHOLESTEROL WITH WATER AND A REAGENT CONTAINING ACETIC ACID, ACETICANHYDRIDE AND SULFURIC ACID TO FORM A COLORED SUBSTANCE WHICH ABSORBSLIGHT AT 500 NM, AND MEASURING THE OPTICAL DENSITY OF THE COLOUREDSUBSTANCE.
 2. In a method for determining the amount of cholesterol inblood serum in which blood serum is treated with a reagent comprisingsulfuric acid, acetic acid and acetic anhydride to form a coloredproduct and the intensity of the color produced is measured to give avalue which is proportional to the concentration of the cholesterol inthe blood serum, wherein the improvement comprises diluting the bloodserum with water and maintaining the ratio of blood serum to water toreagent within the range of one unit volume of blood serum to 15-24 unitvolumes of water to 200 unit volumes of reagent.
 3. The method as setforth in claim 2 including the step of measuring the absorbance of thecolor produced formed at 500 nm.
 4. The method as set forth in claim 3wherein the ratio of serum to water to reagent is 1 to 19 to
 200. 5. Amethod for determining the amount of cholesterol in blood serumcomprising diluting the blood serum with water, treating the blood serumwith a reagent comprising sulfuric acid, acetic acid and aceticanhydride to form a colored substance which absorbs light at 500 nm,thereafter measuring the intensity of the colored substance at 500 nm.6. The method as set forth in claim 5 wherein the ratio of serum towater to reagent is maintained within the range of 1 to 15-24 to
 200. 7.The method as set forth in claim 5 wherein the serum is diluted withwater to have a serum to water ratio of 1 to
 20. 8. The method as setforth in claim 7 wherein 1,000 ml of the reagent contains approximately483 ml of acetic anhydride, 276 ml of acetic acid and 241 ml of sulfuricacid.
 9. The method as set forth in claim 8 wherein each 100 Mu l ofblood serum and water is treated with 1,000 Mu l of reagent.